We examined the effect of overexpression of TLR2 and TLR4 on apoptosis. TLR2 and TLR4 transfected CHO cells were subjected to serum deprivation for 0, 24, and 48 h. CHO cells served as control. The survival was 80.4% and 66.8% in CHO cells, 73.8% and 47.6% in TLR2/CHO, and 70.5% and 53.0% in TLR4/CHO, respectively. Flow cytometry examination suggested that apoptotic cells were 7.17% and 32.91% in control CHO cells, 29.0% and 64.6% in TLR2/CHO, and 41.4% and 64.6% in TLR4/CHO, respectively. The levels of FasL and caspase-8 activity in TLR2/CHO and TLR4/CHO cells were significantly higher than that of CHO cells. Transfection of dominant negative FADD into TLR2/CHO and TLR4/CHO cells significantly reduced apoptosis. Our results suggest that overexpression of TLR2 and TLR4 in CHO cells sensitizes the cells to serum deprivation-induced apoptosis and that the mechanisms are involved in the death receptor-mediated signaling pathway.

Toll-like receptor 4 (TLR4), a key member of the TLR family, has been well characterized by its function in the induction of inflammatory products of innate immunity. However, the involvement of TLR4 in a variety of apoptotic events by an unknown mechanism has been the focus of great interest. Our investigation found that TLR4 promoted apoptotic signalling by affecting the glycogen synthase kinase-3β (GSK-3β) pathway in a serum-deprivation- induced apoptotic paradigm. Serum deprivation induces GSK-3β activation in a pathway that leads to subsequent cell apoptosis. Intriguingly, this apoptotic cascade is amplified in presence of TLR4 but greatly attenuated by β-arrestin 2, another critical molecule implicated in TLR4-mediated immune responses. Our data suggest that the association of β-arrestin 2 with GSK-3β contributes to the stabilization of phospho-GSK-3β, an inactive form of GSK-3β. It becomes a critical determinant for the attenuation of TLR4-initiated apoptosis by β-arrestin 2. Taken together, we demonstrate that the TLR4 possesses the capability of accelerating GSK-3β activation thereby deteriorating serum-deprivation-induced apoptosis; β-arrestin 2 represents an inhibitory effect on the TLR4-mediated apoptotic cascade, through controlling the homeostasis of activation and inactivation of GSK-3β.